Project Summary Our chromosomes are continually bombarded with a variety of insults, resulting in damage that must be repaired. By necessity, cells have evolved mechanisms to detect and repair broken strands of DNA, thereby preventing loss of important genetic information. Double-stranded DNA breaks (DSBs) are a type of damage that led to particularly disastrous outcomes. If not corrected, DSBs can lead to gross chromosomal rearrangements, which are the hallmark of all forms of cancer. Surprisingly, DNA replication is the primary source of DSBs. Homologous recombination (HR) is a highly conserved pathway that cells can use to repair DSBs, and HR is necessary to prevent and repair the damage that arises during DNA replication. When a DSB occurs, the DNA ends are processed to generate 3' single-strand DNA (ssDNA) overhangs. The ssDNA ends then pair with homologous sequence elsewhere in the genome, and the missing DNA sequence is replaced using the homologous DNA as a template for replication. Finally, the replicated intermediate is resolved, regenerating the continuity of the broken DNA. While seemingly simple, HR requires the coordinated action of a complex repertoire of proteins, which are responsible for sensing damage, recruiting essential factors, and processing and repairing the damaged DNA. The consequences of disrupting HR are devastating. For example, mutations in the Rad51 recombinase are embryonic lethal in mice, and mutations in human Rad51 are linked to breast cancers. In addition, defects in BRCA2 account for at least 5% of all breast cancers and also confer a genetic predisposition to ovarian cancer. BRCA2 is thought to help regulate HR, and loss of this regulation may be the reason why this gene is linked to hereditary cancers. Major new discoveries will be necessary to fully understand the mechanistic basis for these outcomes. Our overall research program is focused on understanding how (i) proteins sense and respond to damaged DNA, (ii) how DNA damage is repaired, (iii) how DNA replication can lead to damage, and (iv) how replication and recombination are linked. To help address these problems we have developed unique technologies that allow us to directly visualize hundreds of individual molecules using optical microscopy, which enables us to monitor the spatial and temporal progression of DNA repair and DNA replication in real-time at the single-molecule level. Using this approach we seek to define the fundamental mechanisms that our cells use to replicate and repair DNA, with the long-term goal of understanding how errors during these processes can lead to chromosomal rearrangements.